Brain hypoxia, and the extracellular acidosis and hypercapnia that often accompany it, can lead to changes in the regulation of intracellular pH (pHi). Conversely, changes in pH modulate hypoxic and ischemic injury in the mammalian CNS. It is therefore essential to understand the complex interrelationships among pHi regulation in neurons and astrocytes on the one hand, and hypoxia and/or hypercapnia on the other. This project focuses on how hypoxia and/or hypercapnia (acute or chronic) modulate HCO5 transport and pHi;in CNS neurons and astrocytes, and the role that maturation plays in these responses. Having cloned and developed antibodies for several new Na+-coupled HCO3 transporters, we are now in the position to investigate these important problems with powerful molecular tools?genetic manipulations (knockdown techniques and knockout animals), type-specific antibodies, and single-cell PCR. The latter two we will apply to identified neurons and astrocytes immediately after using digital imaging techniques and pH-sensitive fluorescent dyes to study dynamically pHi physiology. This approach will permit us to determine which molecules are responsible for specific changes in pHj physiology?as induced by hypoxia and/or hypercapnia. The four specific aims are to examine the effect of: (1) Acute hypoxia and/or hypercapnia on the activity of HCO3 transporters. (2) Chronic hypoxia and/or hypercapnia on the expression and activity of HCO3 transporters and carbonic anhydrases (CAs) with which they may be associated. (3) Maturation on the baseline expression and activity of HCO3 transporters and CAs. (4) Maturation on the response of HCO3 and CAs to hypoxia and/or hypercapnia. We will perform the cellular experiments on both cultured and freshly-dissociated hippocampal neurons and astrocytes from mice. The use of out-of-equilibrium solutions in aim (1) will make it possible to change [HCO3]o, pHo and [CO2]o one at a time and determine whether the cells can individually sense each of these parameters. In addition, we will isolate tissues from mice chronically exposed to an environment of hypoxia and/or hypercapnia, and study expression in these tissues using northern and western blotting, PCR and immunocytochemistry. The proposed work should not only clarify how hypoxia and/or hypercapnia?as influenced by maturation?affect pHi regulation in neurons and astrocytes, the work should also clarify at a molecular level how these changes take place.